Chronic hepatitis C virus infection after treatment for pediatric malignancy.

Sera of 658 patients who had completed treatment for pediatric malignancy were analyzed by a second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay test to assess the prevalence of hepatitis C virus (HCV)-seropositivity. All HCV-seropositive patients underwent detailed clinical, laboratory, virologic, and histologic study to analyze the course of HCV infection. One hundred seventeen of the 658 patients (17.8%) were positive for HCV infection markers. Among the 117 anti-HCV+ patients, 41 (35%) were also positive for markers of hepatitis B virus infection with or without delta virus infection markers, 91 (77.8%) had previously received blood product transfusions, and 25 (21.4%) showed a normal alanine aminotransferase (ALT) level during the last 5-year follow-up (11 of them never had abnormal ALT levels). The remaining 92 patients showed ALT levels higher than the upper limit of normal range. Eighty-one of 117 (70%) anti-HCV+ patients were HCV-RNA+, with genotype 1b being present in most patients (54%). In univariate analysis, no risk factor for chronic liver disease was statistically significant. In this study, the prevalence of HCV infection was high in patients who were treated for a childhood malignancy. In about 20% of anti-HCV+ patients, routes other than blood transfusions are to be considered in the epidemiology of HCV infection. After a 14-year median follow-up, chronic liver disease of anti-HCV+ positive patients did not show progression to liver failure.

Immunoblot analyses of chimpanzee sera after infection and after immunization and challenge with Mycoplasma pneumoniae.

Consecutive weekly or biweekly serum specimens obtained during a 3- or 4-month study from 16 chimpanzees were examined by immunoblot analyses to identify the immunogenic components of Mycoplasma pneumoniae. Six experimentally infected chimpanzees showed significant signs of overt disease, including cough, pharyngitis, rhinitis, fever, and loss of appetite. The sera of these infected chimpanzees recognized from 17 to 20 protein bands. Two control chimpanzees that were not inoculated were included in the study. Three chimpanzees immunized with a formalin-inactivated OSU-1A vaccine and three chimpanzees immunized with an experimental acellular vaccine showed minimal signs of disease on challenge. After challenge, the serum immunoblot responses of the immunized chimpanzees were similar to those of the infected chimpanzees. Before challenge, the sera of two previously infected chimpanzees recognized protein bands of 169 (which comigrated with the P1 adhesin), 148, 130, 117, 86, 61, 44, 35, 30, and 29 kDa. After challenge, the previously infected chimpanzees showed the most intense serum immunoblot responses and were most protected against colonization and disease. The sera from each of the 16 chimpanzees examined recognized a large number of immunogenic components, and the serum immunoblot responses were virtually identical to those of patients. Sera from each chimpanzee and patient recognized 169-, 148-, 130-, 117-, 86-, 44-, and 35-kDa bands and many of them recognized 67-, 63-, 61-, 56-, 32-, 30-, and 29-kDa protein bands.

The immunodominant 90-kilodalton protein is localized on the terminal tip structure of Mycoplasma pneumoniae.

Immunoblot analysis of convalescent-phase sera of experimentally infected chimpanzees or monoclonal antibodies (MAbs) specific to the 90- and 40-kDa proteins of Mycoplasma pneumoniae indicated that both proteins were present in cytadsorbing, pathogenic strains PI-1428, M129, and FH but absent in noncytadsorbing, nonpathogenic strain M129-B176. Adsorption of convalescent-phase chimpanzee sera with virulent strain PI-1428 removed reactivity, whereas adsorption with avirulent strain M129-B176 did not remove reactivity to these two proteins. By using proteolysis and specific MAbs, we demonstrated that the 90- and 40-kDa proteins were surface exposed. Immunoelectron microscopy employing specific MAbs showed that the 90-kDa protein is localized on the terminal tip attachment apparatus. However, the MAb specific for the 40-kDa protein failed to indicate a similar localization. Nevertheless, these data, taken together, indicate that the immunodominant 90- and 40-kDa proteins are surface exposed, are localized on the terminal tip apparatus, and might be involved in the attachment mechanism.

Microbial ecosystems as targets of antibiotic actions.

The old representation of the microbe-host interactions as a fight between two contenders has to be considered obsolete. Ecological studies have opened new routes to interpret symbiosis phenomena and to understand in more detail the significance of the microbial communities which are present in various niches of the skin and mucous membranes. In these ecosystems, the resident microorganisms play an important physiological role, frequently with mutualistic relationships. The great stability of these ecosystems is due to the constant composition of microbial communities. One of the main benefits that humans derive from their microbiota is protection from infections. Every change in this biological equilibrium, i.e. modifications of microbial communities (caused by exogenous or endogenous factors) can carry several negative consequences. In this context, exposure to antibiotics (either in the course of therapy or not) can produce direct changes in the target bacterial cells (adhesion, etc.) or modify the whole life of ecosystems by altering the quantitative or qualitative balance within different microbial populations. A selection of antibiotic-resistant organisms can propitiate the genetic transfer of resistance to other autochthonous or allochthonous microorganisms. Multiple-resistant organisms such as those involved in nosocomial infections have amplified the magnitude of this problem. In addition an impairment of host defences can favor infections caused by microorganisms originating from resident communities whose equilibrium has been altered by the antibiotic.

Relevance of ionic effects on norfloxacin uptake by Escherichia coli.

The uptake of the quinolone drug norfloxacin by Escherichia coli was investigated at initial rate kinetics at different pH and monovalent/divalent metal ion concentration. The results support a simple diffusion mechanism for quinolone incorporation into cells. The uptake process decreases under acidic conditions. The presence of Na+ or K+ ions does not affect the results to an appreciable extent, whereas divalent ions cause a dramatic decrease in drug incorporation. The antibacterial activity, evaluated under identical experimental conditions, shows a direct relationship with the uptake data. As a general explanation for the above results it is suggested that the ability of the drug to penetrate into cells is a function of its net charge. The molecule in the zwitterionic form exhibits maximum permeation properties, whereas the uptake is remarkably reduced when the drug bears a net charge as a result of ionization or complex formation with bivalent ions. These results allow further insight into the mechanism of quinolone access to the intracellular compartment.

Synthesis and biological activity of dansyl thymidines.

The synthesis of thymidines selectively dansylated in the 3'- and 5'-positions is reported. The biological investigation showed that these fluorescent nucleosides behave as competitive inhibitors of thymidine kinase (TK) from herpes simplex virus (HSV) and are endowed with a certain degree of antiviral activity against HSV-2 and HSV-1.

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain.

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.

Mechanisms of macrolide resistance in Ureaplasma urealyticum: a study on collection and clinical strains.

Ureaplasma urealyticum is considered as a species which is intrinsically sensitive to macrolides (MIC less than 1 microgram/ml). Nevertheless, some of the strains recently isolated in our laboratories showed moderate to high levels of resistance (MICs ranging from 2 micrograms/ml to 100 micrograms/ml). In particular, a strain (CT28) isolated from a patient with nongonococcal urethritis long treated with erythromycin revealed a MIC greater than 100 micrograms/ml for this antibiotic. In order to investigate the mechanisms of resistance, strain CT28 and ten clinical and laboratory U. urealyticum strains were compared for the sensitivity to six antibiotics including three macrolides. Moreover the amount of macrolide uptake and the specific antibiotic binding to ribosomes were studied. Strain CT28 was resistant to josamycin, erythromycin, roxithromycin, lincomycin and clindamycin but sensitive to minocycline. When compared to a sensitive strain, strain CT28 showed a six-fold reduction in intracellular macrolide influx and accumulation and a reduction in antibiotic binding to ribosomes. The mechanisms implicated in these differences may be important for macrolide resistance in U. urealyticum.

In vitro testing of the antibacterial activity of fosfomycin trometamol against urinary pathogens.

The activity of fosfomycin trometamol (FOT) was compared with that of cotrimoxazole (COT) and norfloxacin (NOR) using urine as medium and 10(7) bacteria as inoculum, conditions as close as those found by the administration of the drugs in vivo during the course of a urinary tract infection. The minimum inhibitory concentrations (MIC) of all antibiotics against 100 strains isolated from urinary tract infections were found to be higher than the breakpoint. Concentrations of FOT corresponding to mean and maximal values found in urine after single dose administration within the 0-48 h interval killed the great majority of strains. COT and NOR, when tested under similar conditions, exhibited an antibacterial activity lower than and equal to that of FOT, respectively. In several strains belonging to different species the frequency of mutation to resistance to 2000 and 1000 micrograms/ml of FOT was very low (greater than 10(-7], whereas it was relatively high (1 x 10(-5) to 1 x 10(-7] for 150 micrograms/ml, the two former and the latter being the respective maximal, mean and minimal values found in urine after administration of a single dose.

DAPI-pUC8 complex: a tool to investigate biological effects of nucleic acid-drug interaction.

A complex consisting of pUC8, a 1.8 Md plasmid, and 4'-6-diamidino-2-phenylindole, a DNA-binding agent, has been performed in vitro under different conditions of ionic strength, and used to transform competent cells. A strong interference with the plasmid-coded activities, related to the P/D ratio where at the DNA-drug complex was formed, was shown to occur. Since the compound does not inhibit the uptake process neither affects plasmid activity once dissociated at high ionic strength, it is likely to be acting from inside the cell while still in the form of a DNA-adduct. This system is proposed as a useful tool to investigate the effects on target genes of drugs endowed with DNA sequence specificity.

Bis-substituted hydroxy-anthracenediones: DNA binding and biological activity.

Three new hydroxy-9,10-anthracenedione derivatives (compounds 1-3 in Figure 1), bearing two charged or polar side chains at positions 2 and 4/5 of the tricyclic system, have been investigated for their DNA binding, cytotoxic and genotoxic activity. The interaction mode with nucleic acids is intercalative for compounds 1 and 2, while external and intercalative binding probably coexist for compound 3. Complexation of the nucleic acid occurs in all cases in a cooperative manner, so that drug binding favours further binding to double helical DNA. The scale of the intrinsic binding constant is discussed in terms of the nature and position of the side chain. Cell growth and DNA synthesis inhibition data match quite well with DNA affinity. Alkaline elution experiments show a correlation between drug cytotoxicity and DNA damage. Our results indicate that the position and number of OH groups, as well as of charged side chains, play an important role in modulating drug affinity for DNA and the consequent biological effects.

A search for potential antitumor agents: biological effects and DNA binding of a series of anthraquinone derivatives.

In an effort to establish a relationship between mechanism of binding and affinity to DNA, cytotoxic activity, and genotoxic activity, we have studied four new anthracenedione derivatives bearing charged side chain groups at various positions of the polycyclic aromatic system. Cytotoxicity, genotoxicity, and thermodynamic DNA binding parameters were shown to be directly related, indicating the polynucleotide as an important target for drug action, rather than a minor, subterminal interacting site. This finding was further supported by the observation of extensive anthraquinone accumulation occurring in the nuclear compartment. The relative binding affinities of the drugs are discussed in terms of nature and position of side-chain substituents. Different binding modes were found: three compounds intercalate into the nucleic acid double helix, and one interacts with the exterior of the macromolecule. The biological results suggest that the mode of complex formation plays a less relevant role than DNA binding efficiency.

On the complex nature of the antiviral activity of coumermycin A1: its interference with the replication of herpes simplex virus type 1.

The mechanism of inhibition of the replication of herpes simplex virus type 1 (HSV-1) by coumermycin A1 (CA1), an inhibitor of bacterial DNA gyrase, has been investigated. Concentrations of antibiotic slightly higher than those needed for 50% inhibition of viral growth were able to inhibit viral DNA synthesis in infected cells. This effect was accompanied by a depressed synthesis of viral polypeptides. Protein synthesis was also inhibited in uninfected cells, especially after long exposure to the drug, but not in a cell-free system. In vitro assays of highly purified HSV-1 DNA polymerase in the presence of the drug, provided evidence that the enzyme was a target of CA1. The viral polymerase was in fact inhibited by the antibiotic to an extent comparable to that of viral DNA synthesis in intact cells. In contrast, DNA polymerase alpha, the enzyme involved in chromosomal DNA replication, was relatively insensitive to CA1. The drug was also shown to bind to protein and to viral and cellular DNA.

Antiviral properties of psoralen derivatives: a biological and physico-chemical investigation.

Two isomeric psoralen derivatives (I and II in Fig. 1) bearing charged side chains, have been tested for activity against Herpes Simplex Virus type 1 (HSV-1) in the absence of u.v. irradiation. Striking differences have been observed both in antiviral and cytotoxic activity for the examined compounds, I being appreciably more effective. Metabolic and biochemical studies, as well as physico-chemical measurements indicate DNA as the major target. The different biological behaviour can be fully explained in terms of a modified affinity of the drugs toward DNA. The molecular basis for these findings probably stems from slightly different intercalation geometries, as shown by chiroptical studies. Comparable binding affinities for viral and cellular DNA fully account for lack of selective toxicity found in vivo. The present approach is proposed as a tool for the investigation of structure-function relationships in drug models.

Different ability of novobiocin and coumermycin A1 to interact with nucleic acids.

The possibility of two structurally related antibiotics, Coumermycin A1 and Novobiocin, to interact with nucleic acids was investigated. Only Coumermycin A1 was able to form complexes with DNA showing an apparent affinity constant comparable to that of the interaction with ribosomal RNA. A binding specificity for A + T complementary and repeating sequences was also exhibited by Coumermycin A1. In view of the different behaviour of the two compounds some considerations are made on their mode of action; although they are acting on the same target enzymes in Escherichia coli, they may affect the functions of eukaryotic cells through a different mechanism not equally specific and probably distinct for each of the two antibiotics.